The present invention relates generally to the field of chemistry. In particular, the present invention relates to compositions and methods useful in the field of DNA and RNA synthesis.
Current practice in the field of DNA synthesis employs a procedure comprising the following general steps: (a) sequential assembling of T and variously-protected A, C and G nucleotides on an insoluble solid support; (b) cleavage of the synthesized oligonucleotide from the solid support; and (c) deprotection of the oligonucleotide to produce biologically active material. The past decade has witnessed revolutionary improvements in both the coupling chemistry and in automation of the synthesis process. This has made it possible to assemble a typical oligonucleotide sequence (.sup..about. 20 mer) on a solid support in a matter of one hour. However, complete deprotection of the oligonucleotide has heretofore required treatment at 55.degree. C. for 6 hours when T and the conventional and well established nucleotide derivatives--dA.sup.bz, dC.sup.bz and dG.sup.ibu --are employed. In order to accelerate the deprotection process, the use of protecting groups (for example, phenoxyacetyl groups) which are more labile towards aminolysis by ammonia than those in the conventional nucleotide derivatives has been proposed [Schulhof et al., U.S. Pat. No. 4,980,460; Nucleic Acids Research 15:397 (1987)]. Similarly, relatively labile dimethylformamidine protecting groups have been employed [Vu et al., Tetrahedron Leaders 31:7269 (1990); McBride et al., J. Am. Chem. Soc. 109:2040 (1986)]. Finally, t-butyl phenoxyacetyl groups which are also more labile towards ammonia deprotection have been suggested as an alternative [Sinha et al., Biochimie 75:13 (1993)].
While these types of ammonia-labile protecting groups are useful in reducing the deprotection time to between about 15 minutes and 60 minutes at 55.degree. C., their use is associated with significant drawbacks. First, the lability of the protecting groups translates into a significant amount of instability towards DNA synthesis conditions. In addition, phenoxyacetyl protecting groups reduce the solubility of the nucleotide derivatives in solvents typically employed for oligonucleotide synthesis, such as acetonitrile; as a consequence, in order to use nucleotide precursors comprising these groups a mixture of solvents is necessary. Further, problems have been encountered with these materials with respect to their stability, both on the shelf and during DNA or RNA synthesis. Because of these drawbacks, use of these new nucleotide derivatives has not been widespread and there remains a need for improved compositions and methods for use in the synthesis of DNA and RNA.
The use of N.sup.2 -phenylacetyldeoxyguanosine-5'-phosphate in the synthesis of deoxypolynucleotides has heretofore been proposed [Kohli et al., Indian J. Chem. 18B:272 (1979)]. However, it is reported that removal of the N-protecting group in aqueous pyridine required 2 hours at 50.degree. C. with ammonia. Moreover, N.sup.2 -phenylacetyldeoxy-guanosine-5'-phosphate is not a particularly reactive species in the type of condensation reactions as are conventionally employed in polynucleotide synthesis. Therefore, this compound has not been widely adopted for use in synthesis of polynucleotides.
Similarly, use of the phenylacetyl group for protection of the NH.sub.2 -group in O.sup.6 -alkylguanine has been reported [Li et al., Biochemistry 28:5779 (1989)]. These compounds were phosphorylated to provide the 2-chlorophenyl phosphates or 2-cyanoethyl phosphates for use in oligonucleotide synthesis by the phosphotriester approach in solution. Deprotection of the N.sup.2 -group with aqueous ammonia was reported to have a t.sub.1/2 of at least 48 minutes at 22.degree. C.; the t.sub.1/2 for the alternate deprotection method proposed (oxime/tetramethylguanidine in aqueous ammonia) was at a minimum 1.8 hours.
It is an object of the present invention to provide novel nucleotide derivatives and methods for the preparation and use thereof.